Nitrite and nitric oxide are widespread and abundant in humans and are emerging as a potential new antibacterial therapeutic agents. The oral cavity has particularly high concentrations of nitrite, which can reach 1mM. Oral microorganisms have adapted to survive such high nitrosative stress exposure and disruption of these adaptation mechanisms would be expected to reduce growth and survival of bacteria in the oral environment. Porphyromonas gingivalis, a periodontopathogen, is well known for its high tolerance of nitrosative stress. However, the molecular basis of this tolerance is under-investigated. Using bioinformatics approaches and microarray analysis of P. gingivalis exposed to nitrite and nitric oxide, we identified several potential candidates that may play a role in nitrosative stress protection. The major upregulated gene was hcp, which codes for a putative hydroxylamine reductase. We have further identified a regulator, designated HcpR, which mediates expression of hcp and is required for growth of P. gingivalis in the presence of both nitrite and nitric oxide. We hypothesize that HcpR is a major player in adaptation of P. gingivalis to nitrosative stress. To determine its role in nitrosative stress protection, we will first definethe regulon of HcpR and the minimum DNA sequence required for its binding. Furthermore, we will carry out molecular structure studies of HcpR and of its complexes with DNA using NMR and crystallography. Since our study shows that the major regulated gene in P. gingivalis is hcp, we will determine the role of its gene product in adaptation of the bacterium to nitrosative stress an define its biological function in such adaptation. Also, P. gingivalis codes for multiple players that may be involved in nitrosative stress protection or nitrogen metabolism. We will characterize and determine the roles of those players in adaptation of P. gingivalis to nitrosative stress. Finally, we will investigate the role of nitrosative stress in host-pathogen interactions. The results of our study are expected to provide information regarding nitrosative stress homeostasis mechanisms in P. gingivalis at the regulatory and structural levels This knowledge will provide the tools to design agents that compromise the defense mechanisms of the periodontopathogen and turn endogenous human host nitrite and nitric oxide into a weapon that inhibits growth and ultimately can be exploited to treat periodontal disease. We predict that this work will shed light on nitrosative stress homeostasis mechanisms in a variety of other bacteria that carry similar nitrosative stress protection mechanisms to those in P. gingivalis.